One sixth of the world's population has an infectious disease, and new agents involving novel mechanisms less likely to engender resistance are needed if for no other reason than the almost certain emergence of mutant strains and new pathogens in the future. Porphobilinogen synthase (PBGS;EC 4.2.1.24) is an essential enzyme in the porphyrin biosynthesis pathway. We have found that regulation of the activity of PBGS is dependent upon the assembly state of the protein, in which certain forms are functionally active and others not. PBGS has been shown to exist in a manifold of high-activity octamers and low-activity hexamers whose interconversion is at the level of two different dimer conformations. We predict that regulation of enzyme function by control of nonadditive assembly states in vivo may be common through nature and protein class, termed the morpheein concept. We have identified small-molecules that alter the assembly state of PBGS and inhibit function. For example, "morphlock-1" (MW 413) was selected by computational analysis of the structure of the inactive Pisum sativum (pea) PBGS hexamer because it was predicted to bind in a cavity formed by three of the individual protein subunits. This cavity is present in the inactive hexamer and not in the active octamer. In vitro studies have demonstrated morphlock-1 stabilized the hexamer, causing complete disappearance of the active octameric PBGS, and at the same concentration and conditions effected loss of function. Given the nature of the binding site involved in the protein-protein interface between subunits, we believe it is unlikely that resistance will develop to compounds discovered using this approach. We have also identified three compounds that block the PBGS, essential for life, in the NIAID Category B priority pathogen Yersinia enterocolitica, but not human PBGS, and are active in a Y. enterocolitica zone inhibition assay. These probes provide exciting validation that pathogen-specific regulation of protein assembly state correlates with killing of the pathogen, but are most likely not drug candidates due to their large size and non-proprietary source. Therefore, it is the purpose of this SBIR Phase I grant to use the information we have developed so far, taken together with new insight from X-ray crystallography, if any, and further screening as it emerges, to prepare compound libraries that provide leads and preclinical drug candidates as defined by industry-standard metrics and criteria. The methods that we will use include iterative potency structure activity relationship development, selectivity, and eADME (early absorption, distribution, metabolism, excretion) testing and evaluation. At the end of the second year of this Phase I SBIR, we will have identified 2-3 compounds for preclinical and clinical development in Phase II. In addition, during the course of this work we will also examine possible extension beyond Y. enterocolitica to Plasmodium falciparum, Pseudomonas aeruginosa, Vibrio cholerae, and Toxoplasma gondii. PUBLIC HEALTH RELEVANCE: One sixth of the world's population has an infectious disease and small-molecule therapeutics involving novel mechanisms are needed, especially those less likely to engender resistance. We describe here a new approach for the discovery of anti- infective agents in which species-specific loss of function of an essential enzyme target in a pathogen can be achieved, by regulation of the assembly state of the protein. We first seek to apply our strategy to the discovery of agents to treat Yersinia enterocolitica, an NIAID Category B priority pathogen.